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WO2017152399A1 - Protéine hybride de peptide intestinal vasoactif, procédé de préparation et d'utilisation de celle-ci - Google Patents

Protéine hybride de peptide intestinal vasoactif, procédé de préparation et d'utilisation de celle-ci Download PDF

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Publication number
WO2017152399A1
WO2017152399A1 PCT/CN2016/075992 CN2016075992W WO2017152399A1 WO 2017152399 A1 WO2017152399 A1 WO 2017152399A1 CN 2016075992 W CN2016075992 W CN 2016075992W WO 2017152399 A1 WO2017152399 A1 WO 2017152399A1
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WIPO (PCT)
Prior art keywords
fusion protein
amino acid
acid sequence
vip
vasoactive intestinal
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English (en)
Chinese (zh)
Inventor
李红玉
李加忠
朱红梅
支德娟
李洋
高江利
张丽芸
王亚军
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Lanzhou University
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Lanzhou University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention belongs to the field of genetic engineering pharmacy, and particularly relates to a fusion protein of vasoactive intestinal peptide and a preparation method and application thereof.
  • Vasoactive intestinal peptide is a 28 amino acid polypeptide produced mainly by the peripheral and central nervous systems, released by the parasympathetic ganglia fibers, and coexisted with acetylcholine. A member of the glucagon-trypticoid family. Widely distributed in the choline-like presynaptic nerve cells and peripheral peptide-energy nerve cells of the central nervous system, through which various organs can be innervated, such as the heart, lungs, digestive system and genitourinary tract, eyes, skin, Ovary and thyroid gland.
  • VIP is an important signal peptide in the central and peripheral nervous systems. It plays a physiological role through its receptor. To date, three types of VIP receptors have been cloned and classified, namely VPAC1, VPAC2 and PAC1. Vasoactive intestinal peptides are involved in a variety of biological functions in the body, including participation in metabolism, exocrine and endocrine, cell differentiation and smooth muscle relaxation, hormone secretion, and regulation of immune responses. Based on its many biological functions, VIP is considered to be a drug candidate with therapeutic potential for a variety of diseases including diabetes, asthma, impotence and rheumatism.
  • VIP has a wide range of biological activities, indicating that VIP has broad clinical application prospects. However, only a few clinical trials have been conducted so far. A major obstacle to the clinical application of VIP is that it is subjected to protease action, antibody neutralization, and spontaneous hydrolysis in vivo with a short half-life, which results in a half-life of VIP in the body of less than one minute. In order to extend the half-life of the VIP, it is necessary to improve its stability by modifying and modifying the structure and other methods.
  • Fusion protein technology using human serum albumin (HSA) as a vector is a method of increasing the half-life of proteins.
  • HSA human serum albumin
  • HSA is the main component of human serum, it plays a vital role in the maintenance of plasma volume and osmotic pressure in vivo, and has no enzymatic activity and immunogenicity, good human compatibility, and high molecular weight (about 66KDa).
  • Very low renal clearance, long half-life, about 19 days, high microbial fermentation expression, etc. often used as a transport carrier for in vivo factors and drugs in the body.
  • Increasing the molecular weight of a polypeptide/protein drug by constructing a fusion protein technology can extend the half-life of the drug.
  • HSA can be used as a carrier of small molecule proteins in the blood, thereby prolonging the half-life of small molecule proteins in the human body.
  • Yeh et al. found that the half-life of the HSA-CD4 fusion protein expressed by Kluyveromyces in the rabbit model as an animal model reached 140 times that of the CD4 monomer.
  • the half-life of the HSA-IFN ⁇ fusion protein expressed by W. cerevisiae was 18-fold longer in cynomolgus than in IFN ⁇ monomer.
  • the design of the fusion protein itself is a process with complicated procedures and numerous influencing factors, and the VIP cannot be realized only by the simple addition of the sequence. It is well known to those skilled in the art for the purpose of stabilizing and efficiently expressing and prolonging their half-life.
  • the inventors have disclosed a fusion protein of vasoactive intestinal peptide, the unique amino acid sequence of the fusion protein can ensure high-level stable expression in the host, while retaining the original function of the VIP, the half-life in vivo Significantly extended.
  • Another object of the present invention is to provide a method for producing a fusion protein of vasoactive intestinal peptide.
  • Another object of the present invention is to provide a recombinant expression vector.
  • Another object of the invention is to provide a host expression system.
  • Another object of the present invention is to provide a use of a fusion protein of a vasoactive intestinal peptide.
  • the fusion protein of vasoactive intestinal peptide of the present invention comprises 1 HSA and 1 VIP.
  • the fusion protein package also contains a linker peptide, and the HSA is linked to the VIP via a linker peptide.
  • the fusion protein is prepared by yeast cell expression, wherein the yeast is Pichia pastoris.
  • the VIP is located at the N-terminus of the fusion protein
  • the HSA is located at the C-terminus of the fusion protein
  • the fusion protein is represented by the structural formula as VIP-L1-HSA, wherein L1 represents a linker peptide, and the DNA sequence of L1 is GGCGGTGGCGGCAGCGGTGGCGGC, the amino acid sequence is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly.
  • the VIP has the amino acid sequence shown in SEQ ID NO: 2, the DNA sequence encoding the amino acid sequence of the VIP is as shown in SEQ ID NO: 1; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • the HSA has the amino acid sequence shown in SEQ ID NO: 4, and the DNA sequence encoding the amino acid sequence of the HSA is shown in SEQ ID NO: 3; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • amino acid sequence of the fusion protein is set forth in SEQ ID NO: 6, and the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 5.
  • preparation method of the fusion protein of the vasoactive intestinal peptide comprises the following steps:
  • step 3 The recombinant expression vector described in step 3 is transformed into competent E. coli TOP10, and then transformed into a host expression system for expression, that is, the fusion protein is obtained.
  • the host expression system of step 4 is yeast, and the yeast is Pichia methanolica.
  • the VIP is located at the C-terminus of the fusion protein
  • the HSA is located at the N-terminus of the fusion protein
  • the fusion protein is represented by the structural formula as HSA-L2-VIP, wherein L2 represents a linker peptide, the DNA sequence of L2 is GGTGGTGGCGGCAGC, and the amino acid sequence is Gly- Gly-Gly-Gly-Ser.
  • the VIP has the amino acid sequence shown in SEQ ID NO: 8, and the DNA sequence encoding the amino acid sequence of the VIP is shown in SEQ ID NO: 7; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • the HSA has the amino acid sequence shown in SEQ ID NO: 10, and the DNA sequence encoding the amino acid sequence of the HSA is as shown in SEQ ID NO: 9; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
  • amino acid sequence of the fusion protein is set forth in SEQ ID NO: 12, and the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 11.
  • preparation method of the fusion protein of the vasoactive intestinal peptide comprises the following steps:
  • step (2) 3 The recombinant yeast expression vector described in the step (2) 3 is transformed into competent E. coli TOP10, and then transformed into yeast for expression, that is, the fusion protein is obtained.
  • the yeast is P. methanolophilus.
  • a recombinant expression vector comprising a DNA sequence encoding an amino acid sequence of a fusion protein of a vasoactive intestinal peptide.
  • a host expression system comprising the recombinant expression vector described above.
  • a fusion protein of vasoactive intestinal peptide for preparing anti-inflammatory, anti-injury, cerebrovascular diseases and improving sleep quality.
  • a fusion protein of vasoactive intestinal peptide for use in the preparation of a medicament for treating asthma.
  • the unique amino acid sequence of the fusion protein of the invention can ensure high-level stable expression in the host body, and the half-life of the body is significantly prolonged while retaining the original function of the VIP.
  • yeast extract 5 g of yeast extract, 10 g of peptone, 10 g of NaCl, dissolved in 1000 ml of deionized water, and adjusted to pH 7.0 with 1 mol/L NaOH, autoclaved.
  • Yeast extract 10g peptone 20g, amino acid yeast nitrogen source 13.4g, glycerol 10g, potassium phosphate 26.631g, autoclaved in 1000ml double distilled water, cooled to room temperature, adjusted to pH 6.0, stored at 4 ° C for use.
  • agarose a small amount of ethidium bromide (EB) when it is not hot. Mix it and pour it into it. Put the comb in the glue tank beforehand, cool it until it is completely solidified, and then pull out the comb to use.
  • EB ethidium bromide
  • L29 which is a linker peptide
  • the L1 DNA sequence is GGCGGTGGCGGCAGCGGTGGCGGC
  • the amino acid sequence is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly.
  • PCR was carried out using the DNA of the vector pcDNA3.1-HSA as a template and P1 and P2 as the upstream and downstream primers, respectively.
  • the reaction conditions were as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return step "2", 35 cycles; 72 ° C, 5 min, the total number of cycles is 30 times.
  • the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a DNA band of HSA of about 1.8 kb in size was amplified.
  • the vector p29-simple-VIP, StuI and BspEI were digested, and the corresponding VIP (StuI/BspEI) DNA fragment was recovered.
  • the DNA sequence was as shown in SEQ ID NO: 1, and the amino acid sequence was SEQ ID NO: 2. Shown
  • HSA was digested with PCR-amplified products KpnI and BspEI, and the corresponding HSA (KpnI/BspEI) DNA fragment was recovered by gel, the DNA sequence is shown in SEQ ID NO: 3, and the amino acid sequence is SEQ ID NO: 4. Shown
  • T4 DNA enzyme ligated the VIP (StuI/BspEI) DNA fragment, HSA (KpnI/BspEI) DNA fragment and pPink ⁇ -HC (KpnI/StuI) vector fragment, transformed competent E. coli TOP10, and coated on ampicillin-resistant LB plate 37 Incubate overnight at °C and screen positive clones. The resulting clone was sequenced by Invitrogen, and the clone with the correct sequence was named pPink ⁇ -HC/VIP-L1-HSA.
  • the DNA of the correctly sequenced vector pPink ⁇ -HC/VIP-L1-HSA was digested with AflII to obtain pPink ⁇ -HC/VIP-L1-HSA, which was transformed into yeast competent cells.
  • the transformed bacterial solution was then inoculated on a PAD plate, cultured at 30 ° C for 3-4 days, and positive clones were picked.
  • the positive clones were inoculated into BMGY liquid medium, cultured at 30 ° C for 48 hours, then transferred to BMMY medium to induce expression. After 96 hours, centrifuge at 1500 rpm for 15 minutes, the supernatant was taken, and protein expression was detected by SDS-PAGE electrophoresis.
  • the molecular band of about 70 kD protein is the VIP-L1-HSA fusion protein
  • the amino acid sequence of the fusion protein is SEQ ID NO: 6
  • the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 5. Show. Select the strain with the highest expression level as the engineering strain, and store it at -80 °C for preservation.
  • p29-simple-VIP already contains L2, which is a linker peptide, the L2 DNA sequence is GGTGGTGGCGGCAGC, and the amino acid sequence is Gly-Gly-Gly-Gly-Ser.
  • PCR was carried out using the DNA of the vector pcDNA3.1-HSA as a template and P1 and P2 as the upstream and downstream primers, respectively.
  • the reaction conditions were as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return step "2", 35 cycles; 72 ° C, 5 min, the total number of cycles is 30 times.
  • the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a DNA band of HSA of about 1.8 kb in size was amplified.
  • HSA was digested with PCR-amplified products EcoRI and HindIII, and the corresponding HSA (EcoRI/HindIII) DNA fragment was recovered by gel.
  • the DNA sequence is shown in SEQ ID NO: 9, and the amino acid sequence is as shown in SEQ ID NO: 10.
  • 4T4 DNase was ligated with VIP (EcoRI/XhoI) DNA fragment, HSA (EcoRI/HindIII) DNA fragment and pcDNA3.1 (XhoI/HindIII) vector fragment, transformed into competent E. coli TOP10, and applied to ampicillin-resistant LB plate at 37 ° C overnight. , screening positive clones. The resulting clone was sequenced by Invitrogen, and the clone with the correct sequence was named pcDNA3.1-HSA-L2-VIP.
  • the obtained HSA-L2-VIP DNA fragment has a DNA sequence as shown in SEQ ID NO: 11, and an amino acid sequence as shown in SEQ ID NO: 12.
  • the DNA of the pPink ⁇ -HC (KnI/StuI) vector fragment was recovered by double-cleaving the DNA of the vector pPink ⁇ -HC (product of Invitrogen) with 1KpnI and StuI.
  • the DNA of the correctly sequenced vector pPink ⁇ -HC/HSA-L2-VIP was digested with AflII to obtain pPink ⁇ -HC/HSA-L2-VIP, transformed into yeast competent cells.
  • the transformed bacterial solution was then inoculated on a PAD plate, cultured at 30 ° C for 3-4 days, and clones were picked.
  • the positive clones were inoculated into BMGY liquid medium, cultured at 30 ° C for 48 hours, then transferred to BMMY medium to induce expression. After 96 hours, centrifuge at 1500 rpm for 15 minutes, the supernatant was taken, and protein expression was detected by SDS-PAGE electrophoresis.
  • the molecular band of about 70 kD protein is the HAS-L2-VIP fusion protein
  • the amino acid sequence of the fusion protein is SEQ ID NO: 12
  • the DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: Show.
  • the strain with the highest expression level was selected as the engineering bacteria, and stored at -80 °C for preservation.
  • Example 3 Detection of biological activity of fusion protein of vasoactive intestinal peptide
  • Human colon cancer cell line HT-29 was purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences.
  • a 24-well plate was plated with 2 ⁇ 105 HT-29 cells per well, 1 mL per well. After the cell confluence was 80%, wash once with PBS, add 0.1 mmol/L IBMX, 500 mL per well, and after 30 min of treatment, add The fusion protein and VIP of purified vasoactive intestinal peptide were expressed in Pichia pastoris, 500uL per well. After 30min treatment, the supernatant was aspirated, washed 3 times with cold PBS, finally washed, and then added with appropriate cell lysate. It was frozen in a refrigerator at -20 ° C for 30 min, and slowly thawed at room temperature, and it was allowed to freeze and thaw repeatedly until the cells ruptured. The supernatant was collected by centrifugation at 600 g, 4 ° C, and stored at -20 ° C until use.
  • the biological activity of the above-prepared cell lysate was measured by a competitive ELISA kit: except for the NSB (non-specific binding) well, a primary anti-50 uL was added to each well, and the supernatant was decanted after incubating at 37 ° C for 1 hour. Wash 4 times with Wash Buffer for 1 min each time, then add 50 uL of cAMP Conjugate to each well, add standard solution and corresponding sample in 15 min, 100 uL per well, and incubate at 37 °C for 2 h.
  • NSB non-specific binding
  • the blank control was DMEM/F12
  • the negative control was empty vector (pPink ⁇ -HC) transferred to the supernatant of Pichia methanolica broth
  • the positive control was 1 ⁇ 10 -9 mol/L VIP.
  • the fusion protein of the vasoactive intestinal peptide disclosed by the present invention has higher activity and better anti-inflammatory effect than the positive control group.
  • Thirty wister rats were divided into six groups, about 180-220 g: 1-control group, 2-model group, 3-dexamethasone group, 4-VIP group 1,5-VIP group 2,6-vasoactive intestinal A fusion protein group of peptides.
  • the control group was intraperitoneally injected with 1 mL of normal saline, and the other groups were given 1 mL of physiological saline (containing 2 mg of OVA and 100 mg of aluminum hydroxide) to induce sensitization.
  • the other groups except the control were challenged with 1% OVA once a day for 30 minutes for 7 days; the control group was replaced with physiological saline.
  • the dexamethasone group was intraperitoneally injected with dexamethasone in the first half hour before each nebulization, VIP was injected into VIP group 1 and 2, and the fusion protein of vasoactive intestinal peptide was intraperitoneally injected with vasoactive intestinal peptide.
  • Rats with bronchoalveolar lavage fluid were taken within 24 hours after the last aerosol challenge on the seventh day of dexamethasone group and VIP group.
  • the counts of various inflammatory cells were counted to investigate the changes in cytology after asthma.
  • the blood levels of abdominal aorta were detected by ELISA.
  • the levels of tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin-4 (IL-4) were detected by ELISA.
  • the lung tissue of rats was taken to make pathological specimens of lung tissue to observe the morphology of lung tissue. Variety.
  • VIP group 2 stopped the injection of VIP, and the fusion protein of vasoactive intestinal peptide stopped the injection of vasoactive intestinal peptide fusion protein.
  • the two groups continued to be stimulated with 1% OVA once a day for 30 minutes each time.
  • the rat bronchoalveolar lavage fluid (BALF) was taken within 24 hours after the last atomization challenge.
  • the counts of various inflammatory cells were counted to investigate the changes of cytology after asthma.
  • the abdominal aorta was taken by ELISA.
  • the levels of tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin-4 (IL-4) in serum were changed.
  • the lung tissue of rats was taken to prepare pathological specimens of lung tissue to observe the morphological changes of lung tissue.
  • mice The weight of the normal mice is slower than normal, and it is irritated, sneezing, and tarnished; the breathing is rapid, coughing, forelimb contraction, nodding or abdominal breathing, irregular rhythm, slow movement, asthma The performance, and the symptoms increase with the increase in the number of attacks.
  • the fusion protein group of VIP group 1 and vasoactive intestinal peptide was similar to or similar to the control group.
  • the levels of tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin-4 (IL-4) in serum were detected by ELISA.
  • the levels of 2IL-4 and TNF- ⁇ in the model group and VIP group were significantly higher than those in the control group.
  • the fusion protein group of VIP group 1 and vasoactive intestinal peptide is similar to or close to the control group.
  • the VIP protein has anti-inflammatory, anti-injury, cerebrovascular diseases, and the effect of improving sleep quality
  • the fusion protein of the vasoactive intestinal peptide disclosed in the present invention has the same efficacy, which can be understood by those skilled in the art.
  • the invention discloses a fusion protein of vasoactive intestinal peptide in preparing anti-inflammatory, anti-injury, cerebrovascular diseases and improving sleep The application of sleep quality drugs.
  • the invention discloses a use of a fusion protein of vasoactive intestinal peptide in the preparation of a medicament for treating asthma.
  • the unique amino acid sequence of the fusion protein disclosed in the invention can ensure high-level stable expression in the host, and the half-life of the body is significantly prolonged while retaining the original function of the VIP.

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Abstract

L'invention concerne une protéine hybride d'un peptide intestinal vasoactif, la protéine hybride comprenant une albumine sérique humaine (HAS) et un peptide intestinal vasoactif (VIP). La séquence d'acides aminés unique de la protéine hybride peut assurer qu'elle est exprimée de manière stable à un niveau élevé dans l'hôte, prolongeant ainsi considérablement la période de demi-vie in vivo en même temps, tout en maintenant la fonction d'origine du VIP. En même temps, l'invention concerne un procédé pour préparer la protéine hybride, ainsi qu'une utilisation de celle-ci dans la préparation de médicaments anti-inflammatoires, de médicaments de résistance à des lésions et de médicaments pour traiter des maladies cérébrovasculaires et pour améliorer la qualité du sommeil.
PCT/CN2016/075992 2016-03-09 2016-03-09 Protéine hybride de peptide intestinal vasoactif, procédé de préparation et d'utilisation de celle-ci Ceased WO2017152399A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101875923A (zh) * 2009-12-31 2010-11-03 杭州北斗生物技术有限公司 人血清白蛋白与尿酸氧化酶的融合蛋白及其制备方法
CN102596217A (zh) * 2009-08-14 2012-07-18 费斯生物制药公司 经修饰的血管活性肠肽
WO2012170524A1 (fr) * 2011-06-06 2012-12-13 Phasebio Pharmaceuticals, Inc. Utilisation de peptides intestinaux vasoactifs (piv) modifiés pour traiter l'hypertension
CN102827290A (zh) * 2012-09-07 2012-12-19 浙江大学 人血清白蛋白与人生长激素脂肪分解结构域的融合蛋白

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596217A (zh) * 2009-08-14 2012-07-18 费斯生物制药公司 经修饰的血管活性肠肽
CN101875923A (zh) * 2009-12-31 2010-11-03 杭州北斗生物技术有限公司 人血清白蛋白与尿酸氧化酶的融合蛋白及其制备方法
WO2012170524A1 (fr) * 2011-06-06 2012-12-13 Phasebio Pharmaceuticals, Inc. Utilisation de peptides intestinaux vasoactifs (piv) modifiés pour traiter l'hypertension
CN102827290A (zh) * 2012-09-07 2012-12-19 浙江大学 人血清白蛋白与人生长激素脂肪分解结构域的融合蛋白

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GANEA, D. ET AL.: "The Neuropeptide VIP: Direct Effects on Immune Cells and Involvement in Inflammatory and Autoimmune Diseases", ACTA PHYSIOL., vol. 213, no. 2, 28 February 2015 (2015-02-28), pages 442 - 452, XP055413760 *
SANLIOGLU, A.D. ET AL.: "Therapeutic Potential of VIP vs PACAP in Diabetes", JOURNAL OF MOLECULAR ENDOCRINOLOGY, vol. 49, 18 September 2012 (2012-09-18), pages R157 - R167, XP055413900 *

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